Cholesterol assay

ABSTRACT

Enzyme preparations which will convert cholesterol to Delta 4cholestenone and hydrogen peroxide are obtained from certain Nocardia species belonging to the Mycobacterium rhodocrous group. The preparations have a cholesterol oxidase specific activity of at least 1 unit per 5 mg of protein nitrogen and, when in liquid form, a potency of at least 10 2 units/ml. They are prepared by growing the organism and recovering the enzyme preparation, preferably by extracting the harvested cells with a surface active agent such as Triton X-100. The enzyme preparations are used to assay for cholesterol by measuring the amount in which one of the products of the cholesterol oxidase reaction, preferably hydrogen peroxide, is formed or the quantity in which oxygen is used.

United States Patent 11 1 Richmond 1 Sept. 23, 1975 1 1 CHOLESTEROLASSAY William Richmond, Middlesex, England [75] Inventor:

[73] Assignee: National Research Development Corporation, England 22Filed: Jan. 23, 1974 [21] Appl. No.: 435,892

[30] Foreign Application Priority Data Sept. 22, 1971 United Kingdom44095/71 June 19, 1972 United Kingdom 28650/72 [52] US. Cl. 195/1035 R;195/99 [51] Int. Cl. G0ln 31/14 [58] Field of Search 195/1035 [56]References Cited UNITED STATES PATENTS 3,099,605 7/1963 Free 195/103.5 R3,183,173 5/1965 Oakes 195/103.5 R

3,371,019 2/1968 Hammer et a1. l95/l03.5 R 3,607,093 9/1971 Stone195/1035 R 3,776,816 12/1973 Terada et a1 195/1035 R OTHER PUBLlCATlONSChemical Abstracts, 66, 83303n (1968).

J. Biol. Chem., 206, 511-523 (1954).

.I. Bact., 47, 487-494 (1944).

Bio. Chem. 1., 42. 376-383 (1948).

Schatz et al., J. Bact., Vol. 58, pp. 117-125 (1949). Thoma et al., Sci.Repts. 1st. Super. Sanita. Vol. 1, pp. 326-337 (1961).

Stadtman, Methods in Enzymology, Vol. 1, pp. 678-681 (1955).

Chemical Abstracts, 69, 57563q (1967).

Primary Examiner-Alvin E. Tanenh'oltz [57] ABSTRACT Enzyme preparationswhich will convert cholesterol to A -cholestenone and hydrogen peroxideare obtained from certain Nocardia species belonging to theMycobacterium rhodocrous group. The preparations have a cholesteroloxidase specific activity of at least 1 unit per 5 mg of proteinnitrogen and, when in liquid form, a potency of at least 10' units/m1.They are prepared by growing the organism and recovering the enzymepreparation, preferably by extracting the harvested cells with a surfaceactive agent such as Triton X-lOO. The enzyme preparations are used toassay for cholesterol by measuring the amount in which one of theproducts of the cholesterol oxidase reaction, preferably hydrogenperoxide, is formed or the quantity in which oxygen is used.

46" Claims, No Drawings CHOLESTEROL ASSAY This is a division ofapplication Ser. No. 289,581 filed Sept. l5, 1972.

This invention relates to the assay of cholesterol in liquids andparticularly biological fluids such as serum.

The assay of total cholesterol, in its role as an indicator ofatherosclerosis and incipient coronary heart disease, now constitutesabout 3% of the total number of tests performed in the average clinicalchemistry laboratory. In Britain at the present time this representsabout 1V2 million cholesterol assays per'year.

Chloesterol is currently assayed by the Liebermann- Burchard reactionwhich involves the use highly corrosive and viscous reagents andpresents many obstacles to automation.

It is an object of the present invention to provide an assay forcholesterol which does not have thedisadvan tages of theLiebermann-Burchard reaction and which can be readily automated.

Many Nocardia species are capable of metabolising cholesterol and inparticular Stadtman et al (J. Biol. Chem. (I954) 206 51 l-523) hasdisclosed that a soil Mycobacterium is capable of oxidising cholesterolto N-cholestenone. The so-called cholesterol dehydrogenase responsiblefor this reaction can be obtained in a cell-free form of low activity.The production of this cholesterol dehydrogenase from the same soilMycobacterium as a slightly purer soluble enzyme preparation and thedetermination of the activity of the enzyme is described by Stadtman inMethods in Enzymology (1955) 1 678-681 but the soluble enzyme is stillof low activity and no significantpurification was achieved.

It has now been found that two micro-organisms identified as Nocardiaspecies belonging to the so-called Mycobacterium rhodocrous group havecholesterol oxidase activity, i.e. they convert cholesterol to A-cholestenone and hydrogen peroxide, and that this provides the basis foran enzymic assay for cholesterol which can be readily be automated andwhich obviates the difficulties of the Liebermann-Burchard reaction.

These two micro-organisms are referred to as rough and fsmooth strainsand have been given the numbers NCIB 10554 and NCIB 10555 respectivelyby the National Collection of Industrial Bacteria, Torry ResearchStation, Aberdeen. The micro-organisms have also been deposited with theAgricultural Research Service of the US. Department of Agriculture asthe ARS Culture Collection Investigations Fermentation Laboratory,Peoria, Illinois, U.S.A. where they have been given the numbers NRRL5635 and NRRL 5636.

Full details of the organisms are as follows:

Rough Colony NCIB 10554 (NRRL 5635) Morphology (Nutrient agar 30C)Gram-positive, coryneform organisms. No welldeveloped mycelium, butrudimentary branching present. Coccoid forms appear as the culture ages.Nonmotile.

Colonial morphology (nutrient agar 5 days 30C) Circular, flat, entiredry, opaque, creamy-orange colonies. l.5-3 mm in diameter.

Yeast Dextrose Agar 5 days 30C Circular, raised, entire, opaque, palepink colonies. Dry

crusted surface. 1 mm diameter.

Gelatin Agar Irregular colony edge, raised, dull crusted surface; opaqueoff-white colour.

Egg Yolk Agar plates Irregular colony edge,

opaque, buff-coloured.

Characteristics in liquid culture White surface pellicle, whitefloccular deposite which does not completely disperse on shaking, noturbidity.

raised, dull rough surface,

Physiology llll++ill+ No acid was detectable in peptone water sugars.

Ammonium based sugars Fructose Glucose Sucrose Maltose Glycerol.Sorbitol Trehalose Raffinose Dulcitol Lactose Mannitol Starch ArabinoseSmooth colony NCIB 10555 (NRRL 5636) Morphology (Nutrient agar 30C)Gram-positive coryneform organisms. No welldeveloped mycelium, butrudimentary branching present. Coccoidforms appear as the culture ages.Nonmotile.' I

Colonial morphology (Nutrient agar 5 days 30C) Circular, entire, convex,semi mucoid, opaque creamy off-white colonies, 0.5-2 mm diameter.

Yeast Dextrose agar 5 days 30C Convex, entire, smooth shiny, semimucoid, pale pink colonies.

Characteristics in liquid culture White surface pellicle, whitefloccular. deposit which does not completely disperse on shaking.

Physiology Strict acrobe Gelatin hydrolysis Casein hydrolysis Starchhydrolysis Kovaes oxidase Catalase lfltlUlC' Voges-Proskauer (V-P) testI Methyl Red Deamination of phenylalanine Hippuratc hydrolysis Litmusmilk alkaline Utilisation of compounds as sole carbon sources CitrateLactate Malate Succinate Carbohydrates (acid) Acid was not detectable inpeptone water sugars.

Ammonium based sugars. Fructose Glucose Sucrose. Maltose GlycerolSorhitol 'lrehalose Raftinose Dulcitol Xylose Arahinose Mannitol 1Starch The present invention provdies an enzyme preparation derived fromNocardia species NClB 10554 or NClB 10555 having a cholesteroloxidasespecific activity of at least 1 unit per 5 mg of proteinnitrogen.

The preparation may be in liquid form or in solid, e.g. freeze-driedform When the preparation is in solid form it must be reconstituted'with buffer into a liquid form before it can be used in an assay.

Of the two micro-organisms Nocardia species NClB l0554 (the rou ghstrain) is preferred because of the greater ability of the cells of thisstrain to oxidise eho lesterol to A"-eholestenone and hydrogen peroxide.

The minimum potency of the liquid cholesterol oxidase preparationdepends on the assay method for which it is tobe used. Thus in the caseofa preparation which is to be used for the assay of cholesterol by thefluorimetric determination of hydrogen peroxide pro- 'duced the potencyneed only be relatively small. a potency of 10 units/ml of liquidpreparations being sufficient.

The assay for cholesterol "can also be'carried out by usingnon-fluorimetric methods for the determination of hydrogen peroxide.e.g. colorimetric methods. or by measuring the amount in which A-cholestenone is formed or oxygen is consumed in the chloesteroloxidase: reaction. In this .case the potency ofthe cholesterol .oxidasein the liquid preparation should be at least l0" units/ml. l i iParticularly for use in automated analysis it is preferred that theenzyme preparation have a cholesterol oxidase specific activity of atleast I unitper 50 g protein nitrogen and when made up in liquid formhave a potency of at least 0.5 unit/ml.

When the assay depends on the determination of hydrogen peroxide thepresence ofcatalase in-the enzyme preparation reduces the sensitivity ofthe assay. Gener ally a catalase activity of less than 1071 of thecholesteroloxidase activity i.e. less than l0 units ofcatalasc activityper unit of cholesterol oxidase activity, is tolerable and preferablyfor the preparation should have a catalase activity ofless. than 1'7! ofthe cholesterol oxidase activity, i.e. less than 10 units of catalaseactivity per unit of cholesterol oxidase activity.

The catalase activity of the enzyme preparation dependson the method bywhich it has heen prepared and when the preferred method is used asdescribed below the preparation contains only small catalaseof catalesewhich do not reduce the sensitivity of the assay. How ever ifothermethods are used and the preparation does include catalase this caneither be removed in the purification step or inhibited with a eatalaseinhibitor, such as an azidel'eg. sodium azide.

'As used herein one unit of cholesterol o'xidase activity'is defined asthat activity which will oxidize 1 u'mol (10 mol) of cholesterol toA-cholestenone and -hydrogen peroxide per minute at 30C and-pH7. Oneunit of catalase activity is defined as that activity which will convertl p. mol of hydrogen peroxide to water and oxygen per minute-at 25C andp H7.

In general the enzyme preparations according to the invention areprepared by growing Nr'n'urdiu species NClB 10554 and NClB 10555 andrecovering therefrom an enzyme preparation having a cholesterol oxidasespecific activity of atleast 1 unit per 5 mg of protein nitrogen g Theproduction of the enzyme preparation can generally be divided into thefollowing stages:

1. Growth of'the micro-organism and harvesting of y the cells;

2. Extraction of the cholesterol oxidase activity from the cells; and

3. Purifieationand concentration of the preparation ism is grown in aculture medium comprising glycerol as a carbon source. An example of asuitable medium is:

Yeast Extract 2.0

To avoid precipitation of sparingly soluble salts. the aboveconstituents are dissolved separately and then added to a convenientlylarge volume ofdistilled water in the order shown. The medium is thenmade up to the required volume. The preferred incubation temperature isabout 29C and the optimum pH is between pH 6.0 and pH 7.6. Preferablythe pH is controlled at about pH 6.7.

Increased yields of cells can be obtained by aeration and agitation butthe culture has a tendency to foam and high rates of air flow andagitation can not generally be used because of excessive foaming. An airflow rate of 0.2 v/v/min and an impeller speed of 760 rpm. has givenrapid growth without excessive foaming. An anti-foam such aspolypropylene glycol is preferably also used to control foaming.

If the initial inoculation of micro-organism into the culture medium issmall there may be a lag phase of up to 16 hours. The lag phase is afunction of the size of the inoculum and no lag is obtained if aninoculum greater than 2% (based on the final cell weight) is used.

The total incubation time required is generally about 18 to 24 hours.Under optimum conditions the doubling time ofthe culture is about 2hours and a final cell harvest of at least 25 g (wet weight) per litreof cells (5 g dry weight) can be obtained. The culture can be carriedout on a small scale in 5 litre or 10 litre vessels but excellentresults have also been obtained in larger batches in 100 litre and 1000litre vessels, in the latter case using a 500 litre batch.

Cholesterol oxidase production is growth associated with a lag behindcell growth. The enzyme level continues to increase for at least 2 hoursafter the cells stop growing. Although the enzyme cholestrol oxidase isproduced in the absence of an inducer the yield of enzyme can beincreased by inducing with cholesterol. Small quantities of cholesterolmay be added to the culture as a saturated solution in acetone but forlarger quantities the cholesterol is preferably made up as a slurry inwater and a wetting agent (e.g. Tween 80) and the resulting slurrysonicated to break up the cholesterol particles into a fine suspensionbefore addition to the culture.

As examples of the effect of the inducer 0.2 g/l cholesterol added at acell concentration of 5 g (wet weight')/l give an increase in enzymeproduction by the rough strain NCIB 10554 of the order of 4 fold and L5g/l cholesterol added at the same cell concentration given an increasein enzyme production of the order of fold. n

The cells may be harvested by centrifugation. It can be demonstrated atthis stage that the cells themselves have cholesterol oxidase activitywith a typical specific activity of the cells of the rough" strain beingabout 0.5 p. mols/mg/hour.

Cholesterol oxidase activity can be extracted by disrupting the cellsand subsequently removing cell debris. Electron microscopy has revealedthat the forces encountered in grinding and sonication tend to producefragmentation without secondary damage to the cell wall. In disruptionprocesses giving more efficient release of cholesterol oxidase activitylittle fragmentation occurs but damage to the cell wall is extensive.The ef ficiency of a variety of techniques of cell rupture areillustrated below:

Method 2 Total Activity of Recovered in Recovered as Rupture HomogenateSoluble I-Imyme Grinding with 95"? 8.0"

cardiee Ultrasonieation 28'; 7.0! Acetone Powder 52'6 12.5"! Mickleappafatus 33"? 22.0; LKB Xpress 95" 65.0!

The preferred method of cell rupture is thus the use of an X-press..

Although the cholesterol oxidase activity can be re moved by disruptingthe cells and recovering the enzyme in a cell-free supernatant it hasbeen found that the enzyme can be extracted without disruption of thecells. There are, in fact, indications that the cholesterol oxidase issituated on the surface of the organism and it has been found that itcan be removed with good recovery of activity using a surface activeagent. Nonionic surface" active agents have proved particularly suitableand very good results have been obtained with Triton X-lOO(iso-octylphenoxypolyethoxyethanol containing approximately 10 moles ofethylene oxide).

To remove the cholesterol oxidase the cells are suspended in a solutionof the surface active agent buffered at a suitable pH and the suspensionstirred vigorously at, for example, room temperature. The choles teroloxidase is recovered in the supernatant by removing the cellsjforexample by centrifugation. As the cells are not ruptured'in this processprotein release is low, for example about I00 ag/ml.

In experiments to demonstrate extraction with Triton X-l00 5 g cells(wet weight) ofthe rough" strain were suspended in ml. 0.5M Tris/HCIbuffer at pH 8.0 containing concentrations of l, 3 and 5% Triton X-lOO.The suspension was stirred vigorously at room temperature and 10 mlaliquots were removed at 15 minute intervals. On removal each aliquotwas immediately centrifuged at3'500 rpm. in a Mistral 6L centrifuge. Theextracts were assayed for cholesterol oxidase activity with thefollowing results.

Table l "/1 recovery in supernatan Extraction time mins #g cholesteroloxidised/ ;1.l/l5 mins l "/1 Triton X- l ()0 Thus of the activity of thewhole cells can be recovered at 1% Triton X- and up to 78% at 3% TritonX-l00. The surface active agent will. however. rc main in thecholesterol oxidase solution. In the assay method as described below ithas been found that the presenccof Triton X-l maybe advantageous butthat the optimum concentration in the assay mixture is about 0.25%,higher concentrations being inhibitory. The enzyme preparation shouldthus not have a level of surface active agent greater than that whichproduces an acceptable level in the assay mixture. Thus although themost rapid release of enzyme can be obtained at 3% Triton X-l00 it maybe preferable to work at a lower concentration, e.g. 171 Triton X--l00.It may, however. be necessary to remove the surface active agent. forexample by dialysis or gel filtration, or alternatively by precipitatingthe protein (enzyme) out of the surface active agent solution.

Although the cholesterol oxidase preparation pro duced by extraction ofthe cells with a surface active agent may, in some cases, have specificactivity and a potency which is sufficient to conduct an assay it isalmost always necessary to purify and concentrate the extract before itcan be used practically in a cholesterol assay. lnthe case of afluorimetric determination of hydrogen peroxide the enzyme preparationshould have a potency of at least units/ml to provide a result in ashort enough time for an assay to be useful in practice particularlywhere the analysis is automated the preparation should have a potency ofat least 10 and preferably 0.5 units/ml.

. In the case of a non-fluorimetric assay the prepara-' tion should havea potency of at least l0 units/ml and preferably for automatedanalysisat least 0.5 units/ml.

The preparation may. be prepared with a higher potency for example 5units/ml or more but at higher levels of potency will generally bediluted with buffer before use in a test.

in all cases the preparation must have a cholesterol oxidase specificactivity of at least l unit per 5 mg. protein nitrogen. At higher levelsa protein nitrogen the amount of protein present may make the assaysolution too viscous or interfere in the assay. A specific activity ofat least 1 unit per 50 ,ug protein nitrogen is preferred. A particularlysuitable enzyme preparation has been found to be an aqueous preparationwith a specific activity of 1 unit of cholesterol oxidase activity per28 ug protein nitrogen. a potency of 5 units/ml and containing 3% v/vTriton X-l00. This preparation may be diluted for use in an assay, forexample with 0.01M phosphate buffer, so that for automated analysis itcontains 0.5 units/ml or for manual analysis 0.1 units/ml.

The enzyme preparation having cholesterol oxidase activity need not bein aqueous form and it may be, for example, in the form of afreeze-dried powder. In addition to the preparation ofa solublelyophilised powder. the enzyme can be present as a concentrated,buffered solution or in suspension with ammonium sulphate (with orwithout added buffer).

Although very little protein is extracted with the cholesterol oxidasesome catalase activity is generally present in the surfactant extract.The amount of catalase activity which can be tolerated in the finalenzyme preparation depends upon the assay method to be used, thecatalase activity of the preparation being important where the assayinvolves measuring the amount of H 0 produced. As described in moredetail below some assay methods in which H 0 is determined include theuse of peroxidase and in some cases it is possible to overpower limitedamounts of catalase with peroxidase. In addition it is possible toinhibit any concentration of catalase likely to be found in thesurfactant ex tract with an inhibitor for example sodium azide.

The effect of catalase on the sensitivity of an assay can bedemonstrated as follows:

A catalase solution was prepared by dissolving 20 I ofa crystallinesuspension of catalase in 50 ml 0.05 M phosphate buffer pH 7.0.

0.5 ml of this catalase solution was found to break down 7.04 pLM H O(in a final volume of 2.5 ml) in 2.0 minutes. On this basis the catalaseactivity of the solution can be said to be 7.04 units per ml at pH 7.0and 25C.

Varying amounts of this catalase solution were introduced into acholesterol assay system containing 0.5 units of cholesterol oxidase in2.0 ml and the reduction in sensitivity of the assay observed.

Units catalase added "/1 reduction in sensitivity However, the inclusionof 0,] '/r sodium azide in the reaction mixture completely inhibitedeven the highest level of catalase, thus giving 71 sensitivity even inthe presence of catalase.

Catalast should. however. generally be removed from the preparation orat least reduced in amount. for example by chromatography on DEAEcellulose.

Suitable means for purifying the enzyme preparation obtained by surfaceactive agent extraction and optionally catalase removal include ammoniumsulphate precipitation and/or chromatographic methods and/or reducedpressure evaporation. For example the preparation may be concentrated byammonium sulphate precipitation or polyethylene glycol precipitation,desalted on a Sephadex column and then subjected to ion exchangechromatography. It may be necessary in this step to concentrate theactivity of the preparation by about 15 to 40 fold.

For example concentration and purification may be carried out bysubstrate affinity chromatography. Sephadex' LH-20, prepared by hydroxypropylation of G-25, has both hydrophilic and lipophilic properties andcan be swollen in polar organic solvents, water, or mixtures thereof.LH-ZO may be swollen in ethanol saturated with cholesterol (e.g.approximately 4.0 g 71) and a column may be packed with LH-2O preparedin this fashion. Cholesterol may be uniformly distributed throughout thegel by washing the column with distilled water and finally the columnmay be equilibrated with 0.05M potassium phosphate buffer at pH 7.5.Substantial purification of the enzyme preparation may be achieved bythe use of such a column.

Preferably the enzyme preparation is first subjected to ion-exchangechromatography or substrate affinity chromatography, preferablychromatography as DEAE cellulose. and then further concentrated byultrafiltration or reduced pressure evaporation.

The'invention also provides methods for assaying for cholesterol in aliquid. in particular a biological fluid such as serum or plasma. Theassay methods described are not. however. limited to biological fluidsand can be applied quite generally for determining the amount ofcholesterol present in any industrial or food product or in anyindustrial process where cholesterol assay may be considered necessaryor desirable.

oxygen uptake or the amount in which at least one of the products ifformed. Under suitable standardised conditions it is possible toestimate the amount of cholesterol present from the amount of a productformed The invention provides a method of assaying for cho- 5 or theamount of oxygen used in a given time even ifthe lchtel' l i 11 quidwhich comp ses in a ing he qreaction has not been allowed to go tocompletion. uid with an enzyme preparation capable of oxidising smentioned above. when hydrogen peroxide is estichllestcml and hydmge" Pmated fluorimetrically the liquid enzyme preparation j l g f t 't y 'g Qs f j Pi should have a cholesterol oxidase specific activity of at d YTj t c mmum y pcmxldb least 1 unit per mg of protein nitrogen and apotency pm i of at least units/ml. With an other estimation Theinvention also provides a method of assaying for technique it measuringthe amounyt of hydmgcn pep "l l F fld l x P l f the oxidecolorimetrically, measuring the oxygen uptake, 112: ,txg spgdt s kcig iggsz ifi llglg lgg g fh fmm ls e.g. with an oxygen electrode ormeasuring the amount i c of N-cholestenone produced either directly byits car- ?::h ):1 bonyl absorption or by forming a derivative which is lW l c P" measured colorimetrically, the liquid enzyme preparaucts of lch( |c5tcm| oxlflasc reifcmm fmmcd or h tion should have a cholesteroloxidase specific activity amount in which oxygen is used in thecholesterol oxi- (,fat [east 1 unit per 5 mg f i nitrogen and a dascreuctlm- 2O tency of at least 10" units/ml.

The method according to the invention can be used The assay may, ofcourse. either be carried out as an to assay free or total cholesterolin serum. Cholesterol individual test on a single sample or a pluralityot samis, ot course. never tree in plasma in the true sense as y plesmay be tested on an automated basis. The preit is always combined withphospholipids, proteins and 7s ferred enzyme preparation tor automatedanalysis is triglycerides in soluble lipoprotein complexes. lncorpoy onewith a cholesterol oxidase specilic activity of at ration of a surfaceactive agent in the assay mixture, I

east 1 unit per 50 ,ug of protein nitrogen and a potency however, causesdissolution of these complexes, under of at lea 0 unit/ml gentleconditions, allowing all the free cholesterol to be P f I i t f h denzymically oxidised. The estimation of free cholesre y e amour] O y.mgen P L pmw w duced is measured by a system which comprises achroterol may be useful for screening purposes and may. in I t t I M f dI fact. obviate the need to measure total cholesterol. mogemc wager? orreagen 5 L 0 un erguuig colour change in the presence of hydrogenperoxide.

Cholesterol also occursin serum in the form of esters the mm m of h d tb i m I and, iftotal cholesterol is to be assayed. the cholesterol 2 b ul fgi ig? 1 L edbound in this way must first be brought into a form in ig if: 0:]me.nCd y f 5 9 our 0 which it can be attacked by cholesteroloxidase by sagemc wage or redgen ponifying the esters, for example byreaction with alco- Preferred method the P' P holic potassium hydroxideReacmm with 10 N KOH duced is measured bymeans of quadrivalent-titaniumat 75C effects rapid saponification without coagulaand l Orange whichreact to form a Stable red tion of protein our with hydrogen peroxide(Taurnes & Nordschow To conduct the assay the optionally saponifiedfluid 40 T (1968) f- 6l3l- The ummfm of is then incubated withcholesterol oxidise and the reachydrogen Pemxlde Produced measured bythe tion sity of the colour.

In another preferred method the amount of choles- ('Imh-xn'rn! lu-uluxi'Cholesterol 02 choleht 4 en 3 (me terol resent is determined b measurinthe amount of H 0 4s p y g 2 2 hydrogen peroxide produced by means ofthe reaction is preferably allowed to go to completion. The amount ith4-umin0phcnaz0ne in the presence of excess phe- Of cholesterol presentis determined by measuring the no] and peroxidase the reaction being asfollows:

Thus the preferably saponified extract is reacted with cholesteroloxidase in thepresence of peroxidase. 4- aminophenazone and phenol andthe optical density, is measuredat 510 nm. It can be shown thatthere isa lin- .ear relationship between the AOD (difference inoptical density)between a test solution and a control containing no cholesterol and theamount of cholestenone produced. The cholesterol oxidase. peroxidase. 4-aminophenazone and phenol can be made up intoa single reagent and usedin this form. 7

serum may be added to l .0 ml alcoholic KOH (l N) and incubated at 75Cfor minutes. 0.1 mlof the sa ponifie d extract may then be added to 2.5ml of a cholesterol oxidase/peroxidase/fi-aminophenzone/phenol reagentand themixture incubated at to 37C for 5 minutes and the colour read at5 I0 nm.

In this test a 0.171 solution of cholesterol may produce a turbiditywhen added to the enzyme reagent giving an optical density of about0.05. A 0.1471 solution of cholestenone treated in the same manner mayalso produce a turbidity giving an optical density of about 0.08. Theseconditions would only be encountered in an ex tremely abnormal serumcontaining 1.000 mg /1 cho-i lesterol but the presence of a surfaceactive agent such as 0.1% Triton 'X-l00 inthe enzyme reagent preventsthis turbidity. i.e. neither the substrate nor the product produceduring the reaction a colloidal-suspension which would interfere withspectrophotometry.

Any other suitable reaction of hydrogen peroxide may be used to measurethe amount produced and hence the amount of cholesterol in the serum.Exam ples of'such reactions with the references wherea fullerdescription of the reaction may be found are as follows:

I. 2,6-dichlorophenol indophenol can be used as the oxygen acceptorinstead of 4-aminophenazone in'a coupled peroxidase reaction (Clark; &Timms.

Proc. Assoc. Clin. Biochem. (1968) 5, 61)

2. Hydrogen peroxidecan be reacted with guaiaeum in the presence ofperoxidase to give-a blue product (Morley. Dawson,& Marks 'Proc. Assoc.Clin. Biochem. (1968) 5. 42)

3. lodine is liberatedfrom potassium iodide on reaction with hydrogenperoxide and the liberated iodine can then .be reacted to form apinkcolour with diethyl-p-phenylenediamine Chim. Acta. (1969) 25. 415

4. Iodine is liberated from iodide by reaction with hydrogen peroxideand polyvinyl pyrrolidine is used to shift the absorption of iodine fromthe near u.v. towards the visible where is absorption can be read 5.Under the action of peroxidase hydrogen peroxide oxidises homovanilicacid to a highly fluorescent product in alkaline solution (Protein doesnot interfere with this reaction at a -fold dilution of plasma). Theproduct can be measured fluorimetrieally (Phillips & Elevitch, Amer. J.Clin. Path. (1968) 49, 622) 6. Hydrogen peroxide reacts with iodine inthe 'presence ofa molybdenum (IV) catalyst. [fa known excess ofthiosulphate is used to reduce the iodine as it is produced residualthiosulphate can be titrated coulometrically with iodine. (Simon.Christian & Purdy. Clin. Chem. (1968) I4. 463) (Thompson, Clin.

I I0. *In a typical assay according to this method 0.1 ml of-.Although"measuring the hydrogen peroxide. protozobtain the required asy. For exam pleithe; oxygen uptake in the'cholestero-loxidase-reactionean be mea-' surednusing an-oxygen electrode by thetechnique de-" scribed-by Updike &- Hieks in Nat-urea Lond. (1967)diphenyl hydrazone"or'iso-nicotinic'acid hydrazone. or if a.suffieientlypureenzyme is used in a concentration thahexhibits low absorption-at"24'0 'nm -(or if theen-- zymeis im'mobilisedYth'e cholesterol can beassayed by the increase in absorption at 240 nm due to the Acholestenone formed by enzymic oxidation; i

As mentioned abovecholesterol-assay can either b conducted on anautomated basis where a largea lunrber of assays are conducted insuccession or small num'- .bers of samples can be analysed-individually.The reagents required for the as say will depend upon the parzone. ormore othenreagents required for determining the amount off-i 0 produced.Some of the otherreagents may be'compatible' with-the cholesteroloxidase and may beincorp'oratedinto the enzyme preparation to'give acombin'edrreagent. In the case of assay methods requiringthe presenceofperoxidase this can be inf eluded with the cholesteroloxidase to forrn acombined liquidor freeze dried enzymepreparation. .lntthe easelutedimmediately before use-A reagent such as alcobelie-potassiumhydroxide for saponifyingithe.biological fluid is alsoreq'uiredwhentotal cholesterol is to be. measured but-this will generally be suppliedby theuser as .will buffer and other. reagents required for dilution.

For automated analy theeholesterol oxidase preparation is supplied eithconcentrated or freeze dried form to be dilutedor reconstituted beforeuse. If required by theanalysis the preparation may also contain.per.oxidase. The-other reagents will ;genra-lly be supplied by the userv Where individual tests are to be performed kits of the reagents can besupplied. I

The invention thus also provides a kit for assaying the I I amount ofcholesterol in a liquid comprising in associaat 470 'nm (Wate andMar-bach. Clin. Chem. f 1

tion

i. an enzyme preparation which is derived from N0- cdrdia species NClB10554 or NCIB l0555 and which has a cholesterol oxidase specificactivity of at least 1 unit per 5 mg of protein nitrogen; and

ii.'at least one reagent which is capable of being used in thedetermination of the amount in which a product is formed in thecholesterol oxidase reaction. Component (i) is thus the enzymepreparation generally in a form more concentrated than required in thetest to be diluted with buffer by the user. The enzyme preparation maybe in freeze-dried or in concentrated liquid form. The enzymepreparationmay include per oxidase when this is required for the test or any otherreagent required in the final assay which is compatible with thecholesterol oxidase. The kit will. of course. contain each of thecomponents in the amount required for the same defined number of tests.For example the kit may contain sufficient enzyme and other reagents forsay 12 tests. Each reagent may be supplied in a single amount. eg in abottle. the amount required for an individual test being extracted fromthis single amount.

Alternatively the kit may contain unit doses of the enzyme component,each containing at least 0.05 units of cholesterol oxidase activity.i.e. enough for a single test. For example the unit doses of the enzymepreparation may be provided in freeze-dried or concentrated form inindividual vials to bc reconstituted with buffer 'before each test.

Component (ii) of the kit is. where hydrogen peroxide is to bedetermined colorimetrically, usually an oxygen acceptor such as4-aminophenazone and phenol or xylenol orange and quadrivalent titanium.These other reagents can be supplied in bottles either ready for use orin concentrated form to be diluted by the user.

The user will generally supply the buffer required for dilution, thealkali required for saponification where total cholesterol is to beassayed and any other commonfreagents which may be required such assolvents for extraction or dilution. Where the assay is for totalcholesterol an acid reagent. e.g. dilute HCl or H 50 is required forneutralisation of the reagent used for saponification and this acidreagent may be included in the kit. in cases where N-cholestenone isdetermined directly by its'carbonyl absorption and there is no colourreagent this acid reagent may be the only other component of the kitapart from the enzyme preparation. If required the kit may include oneor more standard cholesterol solutions for standardising the assay.

The invention is illustrated by the following examples although it is tobe understood that they do not limit the invention in any way.

EXAMPLE 1 Production of Cholesterol Oxidase 500 litres of sterile growthmedia were inoculated with 1 litre of a seed culture of Nocardia sp.NClBl0554. The growth media for both seed and production cultures were:

g/litre 4 )Z -I CaCl .2H- 0.0l FeS() .7H. 0.0] K HPO; 2.0 M so..7a,o 0.2Glycerol Yeast Extract The pH is 6.7

wt/litre. 600 g ofcholesterol suspended in 2 litres of a mixtureof'lwecn 80 and water (0.03:1 v/v) was added to the culture. During thegrowth phase the maximum doubling time for growth was about 2 hours.After 24 hours when the cell concentration was about 6-7 g dry wt/litrethe microorganisms were harvested by passage through anintermittent-discharge disc bowl centrifuge at 400 litres/hour.(Alternatively a rotary vacuum precoat filter may be used. Thefermentation time may be reduced by inoculating with a larger seedculture).

The harvested cells were suspended in 0.01 M potassium phosphate bufferpH 7.0 containing 0.5% (v/v) Triton X-l00 at 10C to give a final volumeof litres. After gentle stirring for 2 hours (shorter times may be usedwith only a slight reduction in the amount of en-,

zyme extracted) the extracted cells were removed by passage through atubular bowl centrifuge (model 6P. Sharplcs). The clear supernatantobtained was passed through a column (5 litre capacity) at 5C containingDE-52 cellulose (previously equilibrated with 0.01 M pH. 7.0 potassiumphosphate buffer containing 0.5% Triton X-l00 The cholesterol oxidasepresent and any catalase present was retained by the DE-52 cellu lose.The cholesterol oxidase was released by step-wise elution withincreasing molarities of potassium phosphate buffer pH 7.0 (containing0.5% Triton X-l00). at 5c. Any catalase present remained on the column.

The eluent fractions rich in cholesterol oxidase were furtherconcentrated by ultrafilitration at 5C. using a PM-30 membrane. Theretentate solution obtained (3 litres) had a cholesterol oxidaseactivity of 5.5 umole cholesterol oxidised/ml/min at 37C. The overallyield was about 20%. This may be increased by washing of discardedsolids at the extraction and by collecting a larger fraction at theion-exchange step. The enzyme solution was stored in a liquid form at 5Cwith azide added as a preservative. The solution retained virtuallytotal activity for at least 2 months.

The enzyme preparation had a cholesterol oxidase specific activity ofabout 1 unit per 28 uug protein nitrogen and a potency of about 5units/ml. The preparation contained about'3'/( v/v. Triton X-l00.

EXAMPLE 2 Assay of Free Cholesterol in Serum PRINCIPLE Cholesteroloxidase oxidises cholesterol to A cholestenone with the simultaneousproduction of hydrogen peroxide. The hydrogen peroxide produced ischelated with xylenol orange and quadrivalent titanium. The adsorptionof this red coloured complex is measured at 550 nm.

REAGENTS l. 0.01 M phosphate buffer pH 7.0 containing 0.10

g "/1- sodium azide.

2. Cholesterol oxidase solution 5 units/ml (1 ml oxidises 5 uMcholesterol per minute at pH 7.0 and 30C) contains approx. 3.0 71. v/vTriton X-l00.

3. Working enzyme solution 5 mlcholesterol oxidase solution is added to45 ml of the 0.0] M phosphate buffer.

4..Sulphuric acid 2N 56 ml concentrated sulphuric acid is added todistilled water and diluted to 1 liter.

5. Stock titanium 0.001 M 0.08 g titanium dioxide is placed in a 25 mlconical flask and 0.5 g ammo- V nium sulphate and 2.5 ml concentratedsulphuric acid are added. This mixture is heated on a hot plate untilall of the material is dissolved and is colourless. The solution iscooled and made up to 1 liter with distilled water.

6. Stock xylenol orange 0.001 M 0.76 g xylenol orange is dissolved indistilled water and diluted to one liter.

7. Combined colour reagent Add one volume of stock titanium to 0.5volume of 2N sulphuric acid. mix. Add one volume of stock xylenol orangeand 0.5 g Brij-35 (polyoxyethylene lauryl ether) per 1 liter of reagent.

8. Cholesterol standards containing up to 5.0 mM per EXAMPLE 3 Assay ofTotal Cholesterol in Serum PRINCIPLE Cholesterol is .relcased fromlipoprotein complexes and hydrolysedfrom its esters by alkalinehydrolysis. Following neturalisation of the hydrolysate the cholesterolis enzymically oxidised to A-cholestenone. A-

cholestenone is then extracted from the reaction mixture and itsabsorption at 236 nm is measured. REAGENTS 1. 0.05 M phosphate buffer pH7.0.

2. Cholesterol oxidase Stock solution containing 5.0 units/ml and 3.0 71v/v Triton X-100.

3. Working enzyme 5.0 ml of the cholesterol oxidase stock solution isadded to 45 ml of the 0.05 M

phosphate buffer pH 7.0 (l).

4. Ethanolic KOH HN) A Volucon ampoule (May and Baker Ltd) to make 1liter 1 N KOH is diluted to 100 ml with distilled water and made up to 1liter with absolute alcohol.

5. 0.083 N Hydrochloric Acid containing 0.3 v/v Triton X-100.

6. Cholesterol Standards up to 12.9 mM (500 mg 7( Prepared by dissolvingpure dry cholesterol (BDH Biochemical Standard) in isopropyl alcohol.

7. Cyclohexane METHOD 0.2 ml serum or standard is added to 1.0 mlethanolic KOH and the mixture incubated for minutes at 75C.

in stoppered tubes.

0.1 ml of the saponified extract is now added to 1.0 ml 0.083 N HClcontaining 0.3 "/1 Triton X- l00. The solution is mixed before adding1.0 ml of the working enzyme solution. After addition of the cholesteroloxidase the reaction mixture is incubated at 37C for 10 minutes. i I

5.0 ml ethanolic KOH are now added and the A- cholcstenone formed duringthe reaction is extracted into 3.0 ml cyclohcxane.

Blanks are run in a similar fashion except that the working enzymesolution is not added until after the ad- 1 dltion ol the ethanolic KOH.v

The optical density of an extract is read against its blank at 236 nm.

EXAMPLE 4 Kit Formulation for Assay or Free Cholesterol in v SerumPRINCIPLE Cholesterol oxidase oxidises cholesterol to A cholestenonewith the simultaneous production of hydrogen peroxide. The hydrogenperoxide produced is chelated with xylenol orange and quadrivalenttitanium. The absorption of this red coloured complex is measured at 550nm.

REAGENTS 1. 0.01 M phosphate buffer pH 7.0 containing 0.10

g "/1 sodium azide.

2. Cholesterol oxidase solution 5 units 1 ml oxidises 5 pM cholesterolper minute at pH 7.0 and 30C) containing 3.0 v/v Triton Xl00.

3. Working enzyme solution 5 ml cholesterol oxidase solution is added to45 ml of the 0.01 M phosphate buffer (1 Sulphuric acid 2N 56 mlconcentrated sulphuric acid is added to distilled water and diluted to 1liter.

5. Stock titanium 0.001 M 0.08 g titanium dioxide is placed in a 25 mlconical flask and 0.5 ammonium sulphate and 2.5 ml concentratedsulphuric acid are added. This mixture is heated on a hot plate untilall the material is dissolved and is colourless. The solution is cooledand made up to 1 liter with distilled water.

6. Stock xylenol orange 0.001 M 0.76 g xylenol orange is dissolved indistilled water and diluted to 1 liter.

7. Combined colour reagent One volume of stock titanium is added to 0.5volume of 2N sulphuric acid and mixed. One volume of stock xylenolorange and 0.5 g Brij-35 (polyoxyethylene lauryl either) per liter ofreagent is then added.

8. A cholesterol standard containing up to 3.0 mM

per liter is prepared by dissolving pure dry cholesterol (BDHBiochemical Standard) isopropyl alcohol.

The kit for 20 tests includes twovreagents A and B and a standardcholesterol solution (reagent 8 above) Reagent A mls Cholesterol oxidasesolution prepared as reagent (3) above.

Reagent B 40 mls combined colour reagent prepared as reagent (7) above.

METHOD Add ,ul serum or cholesterol standard to 2.0 ml reagent A andincubate at 37 C for 10 minutes.

Add 1.0 ml reagent B to the reaction mixture and continue incubation at37C for a further 5 minutes.

Blanks are prepared by adding serum or cholesterol standard directly toa mixture of 2 mls reagent A and 1.0 ml reagent B and incubating for 5minutes at 37C.

The optical densities of the test solutions are read against theircorresponding blanks at 550 nm. CALCULATION OF RESULTS mM cholesterolfor lltre test 55" Sumdmd EXAMPLE Kit Formulation for Assay of TotalCholesterol in Serum PRINCIPLE Cholesterol is released from lipoprotcincomplexes and hydrolysed from its esters by alkaline hydrolysis.Following neutralisation of the hydrolysate the cholesterol isenzymically oxidised to A-cholestenone and its absorption at 236 nm ismeasured. REAGENTS l. 0.05 M phosphate buffer pH 7.0

2. Cholesterol oxidase Stock solution containing 5.0 units/ml and 3.0 7rv/v Triton X-l00.

3. Working enzyme 5.0 ml of the cholesterol oxidase stock solution isadded to 45 ml of the 0.05 M phosphate buffer pH 7.0 (1).

4. Ethanolic KOH (1N) Volucon" ampoule (May & Baker Limited) to make 1liter 1N KOH is diluted to 100 ml with distilled water and made up to 1liter with Absolute Alcohol.

5. 0.083 N Hydrochloric Acid containing 0.3 7! v/v Triton X-l00.

6. Cholesterol Standard up to 12.9 mM (500 mg "/1) prepared bydissolving pure dry cholesterol (BDH Biochemical Standard) in isopropylalcohol.

7. Cyclohexane.

A kit for tests includes two reagents A and B and standard Cholesterolsolutions (reagent 6 above) Reagent A Cholesterol oxidase 80 ml workingenzyme (reagent 3).

Reagent B 80 mls. 0.083 N hydrochloric Acid containing 0.3 7! v/v TritonX-100.

Ethanolic KOH and cyclohexane are provided by the user. as the former isunstable and both are required in relatively large volumes.

METHOD 0.2 ml serum or standard is added to 1.0 ml ethanolic KOH and themixtue incubated for 5 minutes at 75 in stoppered tubes.

0.] ml of the saponified extract is now added to 1.0 ml 0.083 N HClcontaining 0.3 "/1 Triton X-1OO (Reagent A). The solution is mixedbefore adding 1.0 of the working enzyme solution (Reagent B). Afteraddition of the cholesterol oxidase the reaction mixture is incubated at37 for 10 minutes.

5.0 ml ethanolic KOH are now added and the A- cholestenone formed duringthe reaction is extracted into 4.0 ml eyclohexane.

Blanks are run in a similar fashion except that the working enzymesolution is not added until after the addition of the ethanolic KOH. i

The optical density of the extracts is read against their blanks at 236nm.

1 claim: A

l. A method of assaying for cholesterol in a liquid which comprisesincubating said liquid with an enzyme preparation capable of oxidisingcholesterol into A- cholestenone and hydrogen peroxide and determiningthe amount of cholesterol present in said liquid by measuring the amountof hydrogen peroxide produced.

2. A method according to claim 1 in which said liquid is serum oranother biological fluid.

3.'A method of assaying for cholesterol in a liquid which comprisesincubating said liquid with an enzyme preparation derived from Nocadiaspecies NCIB 10554 (NRRL 5635) or NClB 10555 (NRRL 5636). said enzymepreparation having cholesterol oxidase activity capable of oxidizingcholesterol to A" eholeste none and hydrogen peroxide and determiningthe amount ofcholesterol present in said liquid by measuring the amountin which a product of the cholesterol oxidase reaction is formed or theamount in which oxygen is used in the cholesterol oxidase reaction.

4. A method according to claim 1 and wherein said enzyme preparation isderived from a Nucurdia species.

5. A method according to claim 4 in which said enzyme preparation has acholesterol oxidase specific activity of at least 1 unit per 5 mg ofprotein nitrogen and a potency of at least 10' units ml when in liquidform and the amount of hydrogen peroxide'produced is measured by afluorimetric method.

6. A method according to claim 4 in which said enzyme preparation has acholesterol oxidase specificactivity of at least 1 unit per 5 mg ofprotein nitrogen and a potency of at least 10' units/ml when in liquidform and the amount of hydrogen peroxide produced is measuredcolorimetrically.

7. A method according to claim 4 in which the amount ofhydrogen peroxideproduced is measured by a system which comprises a chromogenic reagentor reagents capable of undergoing a colour change in the presence ofhydrogen peroxide. said amount of hydrogen peroxide being measured bycolorimetrically measuring the colour change ofthe chromogenic reagentor reagents.

8. A method according to claim 7 in which said liquid is serum oranother biological fluid.

9. A method according to claim 8 in which saponified serum or otherbiological fluid is incubated with said enzyme preparation to measuretotal cholesterol.

10. A method according to claim 3 in which said enzyme preparation has acholesterol oxidase specific activity of at least 1 unit per 50 pg ofprotein nitrogen and a potency of at least 0.5 unit ml when in liquidform.

11. A method according to claim 3 carried out as an individual test on asingle sample.

12. A method according to claim 3 in which a plurality of samples aretested on an automated basis.

13. A kit for use in assaying for cholesterol in a liquid comprising inassociation a. an enzyme preparation which is derived from N0- cardiaspecies NClB 10554 (NRRL 5635) or NClB 10555 (NRRL 5636), which has acholesterol oxidase specific activity of at least 1 unit per 5 mg ofprotein nitrogen and is capable of oxidizing cholesterol into 66-cholestenone and hydrogen peroxide; and

b. at least one reagent which is capable of being used in thedetermination of the amount in which hydro gen peroxide is formed in thecholesterol oxidase reaction.

14. A kit according to claim 13 in which component (b) comprises atleast one reagent capable of taking part in a reaction by means ofwhichhydrogen peroxide can be determined fluorimetrically.

15. A kit according to claim 13 which includes in ad dition at least onestandard cholesterol solution.

16. A kit for use in assaying for cholesterol in a liquid comprising inassociation (a) an enzyme preparation capable of oxidizing cholesterolto A-cholestcnone and hydrogen peroxide; and (b) at least one reagentwhich is capable ofbeinguscd in the determination of the amount ofhydrogen peroxide produced.

l7.'A Kit according to claim 16 wherein said-enzyme preparation isderived from a Nm'ardiu species.

18. A kit according to claim 1.7 in which enzyme preparation (a) is inliquid form and has a potency of at least 10 units/ml.

19. Akit according to claim 17 in which component (b) comprises at leastone reagent capable of taking part in a reaction by means of whichhydrogen peroxide can be determined colorimetrically.

20. A kit according to claim 17 in which said enzyme preparation (a) hasa cholesterol oxidase specifieactivity of at least I unit per 50 pg ofprotein nitrogen and when in liquid form. has a potency of at least 0.5unit/ml.

21. A kit according to claim 17 in which component (a) is a freeze-driedenzyme preparation which upon addition of an aqueous solvent and asuitable buffer forms an aqueous preparation of the required potency.

22. A kit according to claim 17 which contains each component in anamount required for a defined number of tests.

23. A kit according to claim 22 which contains a single amount of eachcomponent, the amount of each component required for an individual testto be extractcd from this single amount. I i

24. A kit according to claim 22 which contains a separate amount ofenzyme component (a) for each test.

25. A kit according to claim 24. in which separate amounts of enzymecomponent (a) which correspond to that amount required for a single test'are provided in freeze dried or concentrated form in individual vialsto be reconstituted with buffer before each test.

26. A kit according to claim 13 for the assay of total cholesterol inserum which contains in addition to components (a) and (b) at least oneacid reagent capable of neutralising saponified serum.

27. A method of assaying for cholesterol in a liquid which comprisesincubating said liquid with a cholesterol'oxidase capable of oxidizingcholesterol to A cholestenone and hydrogen peroxide and having a potencyof at least 10 units/ml and determining the amount of cholesterol bymeasuring the amount in which A cholestenone is formed or oxygen isconsumed in the cholesterol oxidase reaction.

28. A kit for use in assaying for cholesterol in a liquid comprising inassociation a. a cholesterol oxidasecapable of oxidizing cholesterol toA cholest enone and hydrogen peroxide and having a potency of at least10 units/ml; and b. at least one reagent which is capable of being usedin the determination of the amount in which A*- LII cholestenone isformed or oxygen is consumed in the cholesterol oxidase reaction.

29. A method according to claim 27 wherein the cholesterol oxidase has aspecific activity of at least unit per 50 pg of protein nitrogen.

30. A kit according to claim 28 wherein the cholesterol oxidase has aspecific activity of at least 1 unit per 50 ,ug of protein nitrogen.

31. A method according to claim 1 wherein the enzyme preparation has acatalase activity of less than l0)? of the cholesterol oxidase activity.

32. A kit according to claim 16 wherein the enzyme 1 preparation has acatalase activity of less than l0? ofthe cholesterol oxidase activity.

33. A method according to claim 1 wherein :the enzyme preparation has acatalaseactivity of less than .i g

of the cholesterol oxidase activity.

34. A kit according to claim 16 wherein the enzyme: I

preparation has a catalase activity of less-than 1;?! of the cholesteroloxidase activity. v

35. A method accordingto claim 4 wherein the enzyme preparation has acholesterol oxidase specific activity of atleast 1 unit per 50 ,ug ofprotein nitrogen and a potency of at least 0.5 units/ml. I v

36. A kit according to claim. 17 wherein the enzyme preparation has acholesterol oxidase specific activity of at least 1 unit per 50 pg ofprotein nitrogen and a potency of at least 0.5 units/ml.

37. A method according to claim 4 wherein the enzyme preparation has acholesterol oxidase specific activity of at least 1 unit per 28 ,ug ofprotein nitrogen, 11

potency of 5 units/ml and contains 371 v/v Triton X- a potency of atleast 10 units/ml when in liquid form and theamount of hydrogen peroxideproduced is measured a fluorimetric method. I

41. A method according to claim 1 in which said enzyme preparation has acholesterol oxidase specificactivity of at least I unit per 5 mg ofprotein nitrogen and a potency of at least l0" units/ml when in liquidform and the amount of hydrogen peroxidepr'oduced is measuredcolorimetrical ly I 1 42. A kit according to claim l6'in which enzymepreparation (a) is in liquid form and has a potency of 'at' least 10"units/ml.

43. A kit according to claim 16 in which the at least one reagent ofcomponent (b) is capable of taking part 1 in a reaction by means ofwhich hydrogen peroxide can be determined colorimetrically.

44. A kit according to claim 16 in which component (a) is a freeze-driedenzyme preparation which upon addition of an aqueous solvent and asuitable buffer forms an aqueous preparation of the required potency.

45.-A' kit according to claim 28 wherein saidcholesterolfoxidase isderived from a Nucardia species.

46. A kit according to claim 28 wherein said cholesterol oxidase isderived from Nucardia species NCIB l0554 (NRRL 5635) or NClB 10555 (NRRL5636).

1. A METHOD OF ASSAYING FOR CHOLESTEROL IN A LIQUID WHICH 2 COMPRISESINCUBATING SAID LIQUID WITH AN ENZYME PREPARATION CAPABLE OF OXIDISINGCHOLESTEROL INTO $4-CHOLESTENONE AND HYDROGEN PEROXIDE AND DETERMININGTHE AMOUNT OF CHOLESTEROL PRESENT IN SAID LIQUID BY MEASURING THE AMOUNTOF HYDROGEN PEROXIDE PRODUCT.
 2. A method according to claim 1 in whichsaid liquid is serum or another biological fluid.
 3. A method ofassaying for cholesterol in a liquid which comprises incubating saidliquid with an enzyme preparation derived from Norcadia species NCIB10554 (NRRL 5635) or NCIB 10555 (NRRL 5636), said enzyme preparationhaving cholesterol oxidase activity capable of oxidizing cholesterol toDelta 4 cholestenone and hydrogen peroxide and determining the amount ofcholesterol present in said liquid by measuring the amount in which aproduct of the cholesterol oxidase reaction is formed or the amount inwhich oxygen is used in the cholesterol oxidase reaction.
 4. A methodaccording to claim 1 and wherein said enzyme preparation is derived froma Nocardia species.
 5. A method according to claim 4 in which saidenzyme preparation has a cholesterol oxidase specific activity of atleast 1 unit per 5 mg of protein nitrogen and a potency of at least 10 2units ml when in liquid form and the amount of hydrogen peroxideproduced is measured by a fluorimetric method.
 6. A method according toclaim 4 in which said enzyme preparation has a cholesterol oxidasespecific-activity of at least 1 unit per 5 mg of protein nitrogen and apotency of at least 10 1 units/ml when in liquid form and the amount ofhydrogen peroxide produced is measured colorimetrically.
 7. A methodaccording to claim 4 in which the amount of hydrogen peroxide producedis measured by a system which comprises a chromogenic reagent orreagents capable of undergoing a colour change in the presence ofhydrogen peroxide, said amount of hydrogen peroxide being measured bycolorimetrically measuring the colour change of the chromogenic reagentor reagents.
 8. A method according to claim 7 in which said liquid isserum or another biological fluid.
 9. A method according to claim 8 inwhich saponified serum or other biological fluid is incubated with saidenzyme preparation to measure total cholesterol.
 10. A method accordingto claim 3 in which said enzyme preparation has a cholesterol oxidasespecific activity of at least 1 unit per 50 Mu g of protein nitrogen anda potency of at least 0.5 unit ml when in liquid form.
 11. A methodaccording to claim 3 carried out as an individual test on a singlesample.
 12. A method according to claim 3 in which a plurality ofsamples are tested on an automated basis.
 13. A kit for use in assayingfor cholesterol in a liquid comprising in association a. an enzymepreparation which is derived from Nocardia species NCIB 10554 (NRRL5635) or NCIB 10555 (NRRL 5636), which has a cholesterol oxidasespecific activity of at least 1 unit per 5 mg of protein nitrogen and iscapable of oxidizing cholesterol into 66 4-cholestenone and hydrogenperoxide; and b. at least one reagent which is capable of being used inthe determination of the amount in which hydrogen peroxide is formed inthe cholesterol oxidase reaction.
 14. A kit according to claim 13 inwhich component (b) comprises at least one reagent capable of takingpart in a reaction by means of which hydrogen peroxide can be determinedfluorimetrically.
 15. A kit according to claim 13 which includes inaddition at least one standard cholesterol solution.
 16. A kit for usein assaying for cholesterol in a liquid comprising in association (a) anenzyme preparation capable of oxidizing cholesterol to Delta4-cholestenone and hydrogen peroxide; and (b) at least one reagent whichis capable of being used in the determination of the amount of hydrogenperoxide produced.
 17. A Kit according to claim 16 wherein said enzymepreparation is derived from a Nocardia species.
 18. A kit according toclaim 17 in which enzyme preparation (a) is in liquid form and has apotency of at least 10 2 units/ml.
 19. A kit according to claim 17 inwhich component (b) comprises at least one reagent capable of takingpart in a reaction by means of which hydrogen peroxide can be determinedcolorimetrically.
 20. A kit according to claim 17 in which said enzymepreparation (a) has a cholesterol oxidase specific activity of at least1 unit per 50 Mu g of protein nitrogen and when in liquid form, has apotency oF at least 0.5 unit/ml.
 21. A kit according to claim 17 inwhich component (a) is a freeze-dried enzyme preparation which uponaddition of an aqueous solvent and a suitable buffer forms an aqueouspreparation of the required potency.
 22. A kit according to claim 17which contains each component in an amount required for a defined numberof tests.
 23. A kit according to claim 22 which contains a single amountof each component, the amount of each component required for anindividual test to be extracted from this single amount.
 24. A kitaccording to claim 22 which contains a separate amount of enzymecomponent (a) for each test.
 25. A kit according to claim 24, in whichseparate amounts of enzyme component (a) which correspond to that amountrequired for a single test are provided in freeze dried or concentratedform in individual vials to be reconstituted with buffer before eachtest.
 26. A kit according to claim 13 for the assay of total cholesterolin serum which contains in addition to components (a) and (b) at leastone acid reagent capable of neutralising saponified serum.
 27. A methodof assaying for cholesterol in a liquid which comprises incubating saidliquid with a cholesterol oxidase capable of oxidizing cholesterol toDelta 4 cholestenone and hydrogen peroxide and having a potency of atleast 10 1 units/ml and determining the amount of cholesterol bymeasuring the amount in which Delta 4-cholestenone is formed or oxygenis consumed in the cholesterol oxidase reaction.
 28. A kit for use inassaying for cholesterol in a liquid comprising in association a. acholesterol oxidase capable of oxidizing cholesterol to Delta 4cholestenone and hydrogen peroxide and having a potency of at least 10 1units/ml; and b. at least one reagent which is capable of being used inthe determination of the amount in which Delta 4-cholestenone is formedor oxygen is consumed in the cholesterol oxidase reaction.
 29. A methodaccording to claim 27 wherein the cholesterol oxidase has a specificactivity of at least 1 unit per 50 Mu g of protein nitrogen.
 30. A kitaccording to claim 28 wherein the cholesterol oxidase has a specificactivity of at least 1 unit per 50 Mu g of protein nitrogen.
 31. Amethod according to claim 1 wherein the enzyme preparation has acatalase activity of less than 10% of the cholesterol oxidase activity.32. A kit according to claim 16 wherein the enzyme preparation has acatalase activity of less than 10% of the cholesterol oxidase activity.33. A method according to claim 1 wherein the enzyme preparation has acatalase activity of less than 1% of the cholesterol oxidase activity.34. A kit according to claim 16 wherein the enzyme preparation has acatalase activity of less than 1% of the cholesterol oxidase activity.35. A method according to claim 4 wherein the enzyme preparation has acholesterol oxidase specific activity of at least 1 unit per 50 Mu g ofprotein nitrogen and a potency of at least 0.5 units/ml.
 36. A kitaccording to claim 17 wherein the enzyme preparation has a cholesteroloxidase specific activity of at least 1 unit per 50 Mu g of proteinnitrogen and a potency of at least 0.5 units/ml.
 37. A method accordingto claim 4 wherein the enzyme preparation has a cholesterol oxidasespecific activity of at least 1 unit per 28 Mu g of protein nitrogen, apotency of 5 units/ml and contains 3% v/v Triton X-100.
 38. A kitaccording to claim 17 wherein the enzyme preparation has a cholesteroloxidase specific activity of 1 unit per 28 Mu g of protein nitrogen, apotency of 5 units/ml and contains 3% v/v Triton X-100.
 39. A methodaccording to claim 2 in which saponified serum or other biological fluidis incubated with said enzyme preparation to measure total cholesterol.40. A method according to claim 1 in which said enzyme preparation has acholesterol oxidase specific activity of at least 1 unit per 5 mg ofprotein nitrogen and a potency of at least 10 2 units/ml when in liquidform and the amount of hydrogen peroxide produced is measured by afluorimetric method.
 41. A method according to claim 1 in which saidenzyme preparation has a cholesterol oxidase specific activity of atleast 1 unit per 5 mg of protein nitrogen and a potency of at least 10 1units/ml when in liquid form and the amount of hydrogen peroxideproduced is measured colorimetrically.
 42. A kit according to claim 16in which enzyme preparation (a) is in liquid form and has a potency ofat least 10 2 units/ml.
 43. A kit according to claim 16 in which the atleast one reagent of component (b) is capable of taking part in areaction by means of which hydrogen peroxide can be determinedcolorimetrically.
 44. A kit according to claim 16 in which component (a)is a freeze-dried enzyme preparation which upon addition of an aqueoussolvent and a suitable buffer forms an aqueous preparation of therequired potency.
 45. A kit according to claim 28 wherein saidcholesterol oxidase is derived from a Nocardia species.
 46. A kitaccording to claim 28 wherein said cholesterol oxidase is derived fromNocardia species NCIB 10554 (NRRL 5635) or NCIB 10555 (NRRL 5636).